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Confirmation of proper plasmid assembly is a critical step to ensuring an HDR donor plasmid has been properly assembled and is viable for use as a DNA donor. The Edit-R HDR DNA Donor Plasmid Colony PCR Primer Pairs are essential components of the Edit-R HDR Plasmid Donor Kit and are utilized to confirm that the 5′ and 3′ homology arms are properly inserted in the completed donor template plasmid. Following assembly of the EGFP or mKate2 DNA donor plasmid from the backbone, insert, and two homology arms; a PCR reaction is carried out across the two homology arms from the backbone to the insert region to ensure proper assembly and orientation. To confirm assembly of a donor plasmid contining a custom sequence, a single PCR reaction amplies the entire region encompassing both homology arms and the insert.

Highlights

Fast and efficient assembly of the DNA donor plasmid

Validated protocols for design, assembly, and confirmation of the donor plasmid

Materials required for cloning HDR plasmid donors

The Edit-R HDR plasmid donor is created through plasmid cloning and assembly of a fluorescent protein insert sequence, the cut vector backbone, and two homology arms generated with custom PCR primers to geneomc regions flanking the insertion site.